Proton-Binding Capacity of Staphylococcus aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity

Raja Biswas1, Raul E. Martinez2, Nadine Go¨ hring1, Martin Schlag3, Michaele Josten4, Guoqing Xia1, Florian Hegler2, Cordula Gekeler1, Anne-Kathrin Gleske1, Friedrich Go¨ tz3, Hans-Georg Sahl4, Andreas Kappler2, Andreas Peschel1* 1 Interfaculty Institute of Microbiology and Infection Medicine, Cellular and Molecular Microbiology, University of Tu¨bingen, Tu¨bingen, Germany, 2 Center for Applied Geoscience, Geomicrobiology, University of Tu¨bingen, Tu¨bingen, Germany, 3 Interfaculty Institute of Microbiology and Infection Medicine, Microbial Genetics, University of Tu¨bingen, Tu¨bingen, Germany, 4 Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), Pharmaceutical Microbiology Unit, University of Bonn, Bonn, Germany

Abstract Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most Gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the Gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.

Citation: Biswas R, Martinez RE, Go¨hring N, Schlag M, Josten M, et al. (2012) Proton-Binding Capacity of Staphylococcus aureus Wall Teichoic Acid and Its Role in Controlling Autolysin Activity. PLoS ONE 7(7): e41415. doi:10.1371/journal.pone.0041415 Editor: Vance G. Fowler, Duke University Medical Center, United States of America Received August 8, 2011; Accepted June 26, 2012; Published July 23, 2012 Copyright: ß 2012 Biswas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the German Research Foundation grants TRR34 to A.P. and F.G. and SFB766 to A.P., G.X., and F.G.; by the Bacteria Materials Interaction graduate programme of the University of Tu¨bingen to A.P. and A.K.; and by a fellowship from the Humboldt Foundation to R.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction impact on the pH-sensitive activity of cell wall-associated enzymes such as autolysins. The bacterial cell envelope governs vital processes including Peptidoglycan is built from long glycan strands that are cross- maintenance of cell shape, cell division, and protection against linked by peptide side chains. Peptidoglycan must be continuously environmental challenges. In addition, it has a crucial role in the synthesized to maintain cell integrity and viability and to allow for respiratory energy metabolism because the cytoplasmic membrane cell division. Peptidoglycan hydrolytic autolysins are critical for harbors the electron transport chain components, which generate separating daughter cells after cell division [11,12]. The mainte- a proton gradient across the membrane that is used to generate nance of the peptidoglycan network requires a fine-tuned spatial ATP or energize transport processes [1,2]. and temporal control of autolysins to prevent suicidal cell death The majority of Gram-positive bacteria contain polyanionic cell [13]. Yet, surprisingly little is known about the control of wall glycopolymers (CWGs) in their cell envelopes, which are autolysins and the underlying regulatory principles are only covalently linked to either peptidoglycan (e.g. wall teichoic acid superficially understood. Inactivation of AtlA, the major autolysin [WTA] or teichuronic acid) or membrane glycolipids (e.g. in S. aureus, or of the corresponding AtlE of Staphylococcus epidermidis lipoteichoic acid [LTA] or succinylated lipoglycans) [3]. Several is not lethal but the mutants form huge cell clusters as a result of roles in the protection and maintenance of Gram-positive cell incomplete cell separation [14]. While studying the role AtlA in S. envelopes and in bacteria-host interaction have been assigned to aureus we observed that a WTA-deficient DtagO mutant (DtagO) CWGs after WTA or LTA-deficient mutants became available in [15] is much more susceptible to autolysis than the wild type when Staphylococcus aureus and Bacillus subtilis [4–6]. Moreover, the cell autolysins were artificially activated by washing the bacteria in envelope of B. subtilis cells has been shown to be protonated during buffer with low ionic strength [16]. We have recently demonstrat- respiratory metabolism [7,8] and the polyanionic CWGs have ed that WTA prevented AtlA binding to peptidoglycan in the been implicated in cation binding [9,10]. However, it has bacterial side walls but not in the septum where WTA appeared to remained unclear if the proton-binding capacity of WTA may be less abundant or to have an altered structure. Similar observations have been made with the cell wall-binding LysM

PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41415 WTA-Mediated Autolysin Regulation domain-containing autolysins LytF of B. subtilis [11] and Sle1 (or WTA affects autolysis via AtlA Aaa) and LytN of S. aureus [17]. Moreover, several studies have The finding that WTA has a strong impact on the abundance of documented that dissipation of the membrane proton gradient protons in the cell wall suggested that the activity of pH-sensitive leads to autolysin activation in Gram-positive bacteria [18,19] but cell-wall associated enzymes such as autolysins might be affected the molecular basis of this phenomenon has remained unclear. by the presence or absence of WTA. In line with this notion WTA Here, we show that WTA contributes substantially to the has been shown to affect the activity of autolysins [20,21]. Activity concentration of proton-binding sites in the cell wall and impacts of AtlA, the major S. aureus autolysin, has its optimal activity at on S. aureus autolysis largely via the major autolysin AtlA. neutral pH and drops strongly at pH values below 6.5 [22] Compounds that modulate the activity of the respiratory chain, suggesting that its activity might be controlled by the presence of a main source of protons in the cell wall, affected autolysis in the WTA. presence of WTA but not in its absence, which supports the notion The autolytic activity of a WTA-deficient mutant DtagO that WTA contributes to the control of autolysin activity by followed a much faster kinetic than that of wild type or governing the abundance of protons in the cell wall. complemented DtagO in the absence of Triton X-100 (Fig. 1) or in its presence as described earlier [20]. In order to analyze if Results WTA deficiency impacts on autolysis via AtlA or via activation of one of the other autolysins atlA and tagO deletions were combined The proton-binding capacity of WTA in the S. aureus cell in strain DtagODatlA. This double mutant exhibited strongly wall reduced autolysis reaching a similar level as in the WTA- We hypothesized that the polyanionic properties of WTA may containing atlA-deficient strain indicating that WTA governs contribute to the binding of protons thereby affecting the local pH autolysis largely via AtlA. in the cell wall. In addition to WTA the cell envelopes of Gram- positive bacteria contain other negatively charged residues in LTA, peptidoglycan, phospholipid head groups, and surface- associated proteins. To assess if WTA contributes significantly to the overall density of negative charges in the cell envelope, the proton-binding capacities of S. aureus wild type and isogenic WTA- deficient tagO mutant (DtagO) were determined by Fourier Transform Infrared (FTIR) spectroscopy and a whole-cell titration approach. Phosphate groups were abundant in wild type cell envelopes but below the FTIR detection limit in DtagO. Accordingly, the proton- binding sites in the mutant were 23% reduced compared to the wild type (Table 1), which confirms that WTA contributes substantially to the proton-binding capacity of the S. aureus cell envelope. DtagO complemented with a plasmid-encoded copy of tagO had the same proton-binding capacity as the wild type indicating that the difference between wild type and DtagO resulted from its inability to synthesize WTA. Since the abundance of protons in the cell envelope might affect the proton gradient across the cytoplasmic membrane the membrane potential of wild type, DtagO, and complemented mutant was measured by monitoring the distribution of the small lipophilic cation [3H]tetraphenylpho- sphonium bromide ([3H]TPP+). The three strains had similar membrane potentials around 2120 mV (Tab. 1) indicating that WTA-dependent proton binding in the cell wall hardly impacts on Figure 1. Increased autolysis of S. aureus DtagO depends on the the abundance of protons at the membrane surface. major autolysin AtlA. AtlA-expressing or deficient strains are indicated by continuous or dotted lines, respectively. Means and SEM of 3 independent experiments are shown. doi:10.1371/journal.pone.0041415.g001 Table 1. Proton-binding sites and phosphate groups in the cell envelope and membrane potential of S. aureus strains.

Proton-binding site Detection of phosphate group Membrane Strain concentrations (mmol/mg bacteria) absorption bands by FTIR (cm21) potential (mV)

Wild type 35.6 1. C-O-P-O-C stretch (1061 cm21)a 2123 2 2. PO2 antisymmetric stretch (1235 cm21)a DtagO 27.5 Bdb 2120 DtagO complemented 35.4 Ndc 2122

a, Assignment was made according to those reported previously in [42]. b, Bd, below detection limit. c, Nd, not determined. doi:10.1371/journal.pone.0041415.t001

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The respiratory chain affects autolysin activity in a WTA- high membrane proton gradient probably causes local acidifica- dependent manner tion (Fig. 3). Since AtlA, the major staphylococcal autolysin, How the respiratory activity affects autolysis in S. aureus has exhibits reduced activity at pH values below 6.5 [22] autolysis may remained elusive. We hypothesized that changes in the membrane remain low during efficient respiratory growth in cell wall areas of proton gradient might lead to local pH changes that govern the high WTA abundance. Thus, WTA appears to control AtlA pH-sensitive activity of AtlA in a WTA-dependent manner. In activity not only by preventing its binding but also by creating an order to elucidate how the respiratory chain affects AtlA activity, acidic milieu that limits AtlA activity. The lower abundance of autolysis was monitored in S. aureus cells whose proton gradient WTA in the septum [16] may help to recruit AtlA to the site of cell was dissipated by addition of the respiratory inhibitor azide with division and activate it by avoiding local acidification. It remains to that of cells exposed to a high concentration of glucose (1%) to be analyzed whether the two ways of how WTA controls autolysin augment respiration. activity are equally important for the control of autolysis in S. In fact, azide led to an increase of autolysis while 1% glucose aureus or other Gram-positive bacteria. In addition to WTA caused a further inhibition of autolysis of S. aureus wild type (Fig. 2), additional mechanisms may contribute to proton gradient- confirming that the respiratory capacity has a considerable impact dependent autolysin regulation in S. aureus. The membrane on S. aureus autolysis. In contrast, the augmented autolysis rate of potential has recently been shown to govern the capacity of the DtagO remained largely unchanged by the addition of azide or LytSR two-component regulatory system to activate the cid and lrg glucose indicating that respiration can only impact on autolysis operons encoding holins and anti-holins that also control autolysin when WTA is present. The complemented mutant behaved like activity [23]. the wild type (Fig. 2). These findings are in agreement with our WTA has many different functions including the targeting and hypothesis that AtlA activity is controlled by the local cell wall pH control of peptidoglycan-biosynthetic proteins [24,25], resistance in dependence of the capacity of WTA to retain respiratory chain- to harmful molecules such as antimicrobial fatty acids [26], derived protons. antimicrobial peptides [27], and lysozyme [28], interaction with inanimate surfaces to initiate biofilm formation [29], binding of Discussion cognate phages [30–32], and interaction with host receptors [33,34]. Its additional role in governing proton binding and We provide evidence that the WTA polymers, which consist of a autolysin activity may explain its widespread occurrence and negatively charged backbone contribute substantially to the usually polyanionic nature. Non phosphate-containing but equally proton-binding capacity of the S. aureus cell wall. WTA is thought anionic cell wall glycopolymers such as the glucuronic acid- to act like a cation exchanger that retains protons in the cell wall containing teichuronic acids found e.g. in bacilli [35], the (Fig. 3). It has been known for long that WTA and the membrane succinylated CWGs of actinobacteria [36], or the pyruvylated proton gradient can affect the autolytic activity of Gram-positive CWGs of Bacillus anthracis [37] may have roles similar to those of bacteria [18,19] but it has remained mysterious if and how these the bona fide teichoic acids. Moreover, it is likely that the phenomena are connected. It has recently been demonstrated that membrane-anchored LTA also contributes to the proton-scaveng- the presence of WTA reduces the affinity of AtlA and of other ing capacities of Gram-positive bacteria [6]. Future studies are autolysins for S. aureus peptidoglycan and that AtlA binds necessary to elucidate if our findings in S. aureus hold true for other preferentially to the cell division site where WTA appears to be Gram-positive bacteria and if WTA or WTA-biosynthetic less abundant or not yet fully matured [16,17]. Accordingly, AtlA enzymes do in fact represent attractive targets for new antibac- is evenly distributed on the surface of DtagO [16], which helps to terial strategies. understand the increased autolysin activity of DtagO compared to the parental strain. However, it does not explain why compounds Materials and Methods that increase or decrease the membrane proton gradient also affect the activity of autolysins. Bacteria, plasmids, and growth conditions Here, we demonstrate that S. aureus autolysis is sensitive to the S. aureus strain SA113 and its derivatives DtagO and DatlA have activity of the respiratory chain in a WTA-dependent manner. A been described recently [16,33]. To construct a DatlADtagO double mutant the complemented DtagO was infected with phage W11 and the mutation was transduced to the DatlA strain. Colonies were screened for erythromycin and spectinomycin resistance with chloramphenicol sensitivity. The correct deletion of tagO in DatlA was confirmed by DNA sequencing. The WTA-deficient DtagO mutant was complemented using plasmid pRB474-SD-tagO, which was constructed by PCR-amplifying tagO from strain SA113 with optimized Shine-Dalgarno sequence and subcloning the amplicon into the pRB474 expression vector [38] to allow tagO expression from its constitutive veg promoter. All strains were grown in B medium (1% casein peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO463H20, 0.1% glucose) containing appro- priate antibiotics when necessary unless otherwise noted.

Figure 2. Autolysis can be increased by addition of azide or FTIR spectroscopy inhibited by addition of glucose in WTA-containing but not in FTIR spectra were recorded using a Bruker Vertex 80v FTIR WTA-deficient S. aureus. Means and SEM of 3 independent spectrometer. The spectral resolution of the spectrometer was set experiments are shown. Between-group differences were analyzed for -1 significance by one-way ANOVA analysis. *, p,0.05; ***, p,0.001; ns, to 2 cm and 256 interferograms with an optical range of 4000 to 21 not significant. 400 cm . To prepare S. aureus wild type and DtagO samples for doi:10.1371/journal.pone.0041415.g002 FTIR measurement, 2 mg of freeze-dried bacterial sample were

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Figure 3. Model for the role of WTA in proton binding and control of autolysin activity. (A) The negatively charged WTA phosphate groups retain protons in the cell wall, which creates an acidic environment keeping the activity of the major autolysin AtlA low. (B) In the absence of WTA protons are not retained, which avoids local acidification and leads to higher activity of AtlA. doi:10.1371/journal.pone.0041415.g003 homogeneously mixed with 250 mg of dry potassium bromide Membrane potentials (99.9%, Sigma-Aldrich) to prepare a KBr pellet. The samples were were determined with the small lipophilic cation [3H]TPP+ freeze-dried in order to remove water for FTIR measurements. whose equilibrium across the cytoplasmic membrane is indicative Slight changes in structure duo to this procedure can, however, of membrane potential as described previously [41]. Briefly, the not be ruled out. test strains were grown in half-concentrated Muller-Hinton broth to an OD600 of 1, harvested, and then resuspended 1:3 in fresh Acid-base titrations and modeling of titration data medium. [3H]TPP+ (25 Ci/mmol; Hartmann Analytic, Braunsch- Bacterial cells were grown until exponential phase and washed weig, Germany) was added to a final concentration of 1 mCi/ml to twice using ultrapure water. Cells were lyophilized and resus- cell suspensions and incubated for 10 min. Subsequently, aliquots pended in 45 ml of 0.01 M NaNO3 background electrolyte at a were filtered onto 0.2-mm-pore-size cellulose acetate membranes concentration of 2 mg/ml for titration experiments. A 40 ml (Schleicher & Schuell, Dassel, Germany) and washed twice with aliquot of the bacterial suspension was then transferred to a 50 mM phosphate buffer (pH 7.0). The filters were dried and 150 ml Metrohm glass titration vessel. Bacterial suspensions were radioactivity was measured using a Packard 1900CA liquid acidified to pH 3 and then covered with an air-tight lid fitted with scintillation counter in the presence of Filtersafe (Zinsser Analytic, an N2 gas line interface to prevent carbonate formation during the Frankfurt, Germany) scintillant. course of the titration and a pH electrode connected to an + autotitrator (Metrohm Titrando 806) to monitor changes in H Autolysis assays activity as a function of added titrant. The titrator settings were Autolysis assays were performed essentially as described recently programmed as described previously [39,40]. [16] with some modification. Briefly, cells were grown until To account for the deprotonation of bacterial cell surface exponential phase, washed twice with PBS at room temperature, functional groups as a function of increasing solution pH the following dissociation mechanism is proposed: HL = L2+H+, and resuspended in prewarmed PBS at an optical density of 0.25 where L2 corresponds to a proton-binding functional group on at 578 nm. Optical density was measured at hourly intervals for + + 3 h at 37uC. Cellular respiration was restored by addition of 1% the cell surface and H ,H3O represents the hydronium ion species in solution. The apparent equilibrium constant for the glucose or inhibited using 50 mM sodium azide as indicated. proton dissociation mechanism described previously, Ka, can be defined as function of measured, [H+], and adjustable, [L2], Acknowledgments + 2 + parameters as follows: Ka = [H ].[L ]/[HL] where [H ] refers to The authors would like to thank Marcus Nowak for the use of his FTIR the proton concentration calculated by the relationship equipment. + + + [H ]={H } cH where, {H } is the proton activity measured using the pH electrode, and cH is the proton activity coefficient for Author Contributions I = 0.01 M (KNO3). A multi-site Langmuir isotherm coupled to a linear programming optimization method (LPM) was used to Conceived and designed the experiments: RB FG HGS AK AP. Performed calculate the concentration of functional groups on the cell surface the experiments: RB RM NG MS MJ GX FH CG AG. Analyzed the data: RB RM MJ. Contributed reagents/materials/analysis tools: MS GX AG. and generate pK (2log K ) spectra where pK is a measure of a a a Wrote the paper: RB AP. functional group acidity as described previously [39].

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